The long-term objectives of my research are to understand the molecular mechanisms that regulate transcription in non-segmented negative-sense (NNS) RNA viruses. These viruses include several significant human, animal and plant pathogens such as the NIAID category A Ebola and Marburg viruses and the category C rabies and Nipah viruses. For many NNS RNA viruses there are no effective vaccines and antiviral drugs, and the development of such therapeutics demands an enhanced understanding of their biology. For decades, vesicular stomatitis virus (VSV) has been studied as a laboratory prototype of all the NNS RNA viruses. The advantages of VSV as a model system include its lack of serious pathogenicity for humans, ability to replicate in a wide range of cultured cells, well established in vitro systems to study RNA synthesis, and a robust reverse genetics system. For these reasons, studies on VSV have frequently provided novel insight into the biology of the less tractable NNS RNA viruses. This proposal aims to understand in mechanistic detail a key stage in viral gene expression, namely how viral mRNA's are processed. Current knowledge in this area indicates that the mechanism by which the viral mRNA's acquire their 5'cap structure is unique, suggesting that these reactions may represent an "Achilles Heel" to which novel broadly active antiviral drugs might be targeted. Using biochemical and genetic approaches we plan to map domains of the RNA polymerase essential for capping and methylation of the viral mRNAs, and determine the substrate requirements for these enzymatic activities. In specific aim 1, we will generate recombinant viruses to test the hypothesis that a transcript must be a minimal length to gain access to the capping machinery. In specific aim 2 we will test the hypothesis that the L protein subunit of the viral polymerase possess guanylyltransferase activity, and identify how the viral mRNA's are recognized for modification. In specific aim 3 we will test the hypothesis that the L protein subunit of polymerase contains two separate methyltransferase domains, and determine how these function to modify mRNA. In specific aim 4 we will test the hypothesis that the 5'mRNA processing events are essential for correct 3'end formation. These experiments should result in the functional assignment of guanylyltransferase and methyltransferase domains within an NNS RNA virus L protein, and thus identify novel targets for the development of antiviral drugs against emerging infectious and potential bio-weapons agents.